Charge Reversal of Polyion Complexes

ABSTRACT

An ionic polymer is utilized in “recharging” (another layer having a different charge) a condensed polynucleotide complex for purposes of nucleic acid delivery to a cell. The resulting recharged complex can be formed with an appropriate amount of positive or negative charge such that the resulting complex has the desired net charge.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation of Application No. 09/328,975, filedJun. 9, 1999, which claims the benefit of U.S. Provisional ApplicationNo. 60/093,153, filed Jul. 17, 1998.

FIELD OF THE INVENTION

The invention relates to compounds and methods for use in biologicsystems. More particularly, polyions are utilized for reversing thecharge (“recharging”) particles, such as molecules, polymers, nucleicacids and genes for delivery to cells.

Background Polymers are used for drug delivery for a variety oftherapeutic purposes. Polymers have also been used in research for thedelivery of nucleic acids (polynucleotides and oligonucleotides) tocells with an eventual goal of providing therapeutic processes. Suchprocesses have been termed gene therapy or anti-sense therapy. One ofthe several methods of nucleic acid delivery to the cells is the use ofDNA-polycation complexes. It has been shown that cationic proteins likehistones and protamines or synthetic polymers like polylysine,polyarginine, polyornithine, DEAE dextran, polybrene, andpolyethylenimine may be effective intracellular delivery agents whilesmall polycations like spermine are ineffective. The following are someprinciples involving the mechanism by which polycations facilitateuptake of DNA:

Polycations provide attachment of DNA to the target cell surface. Thepolymer forms a cross-bridge between the polyanionic nucleic acids andthe polyanionic surfaces of the cells. Polycations protect DNA incomplexes against nuclease degradation. Polycations can also facilitateDNA condensation. The volume which one DNA molecule occupies in acomplex with polycations is drastically lower than the volume of a freeDNA molecule. The size of a DNA/polymer complex is important for genedelivery in vivo.

In terms of intravenous injection, DNA must cross the endothelialbarrier and reach the parenchymal cells of interest. The largestendothelia fenestrae (holes in the endothelial barrier) occur in theliver and have an average diameter of 100 nm. The trans-epithelial poresin other organs are much smaller, for example, muscle endothelium can bedescribed as a structure which has a large number of small pores with aradius of 4 nm, and a very low number of large pores with a radius of20-30 nm. The size of the DNA complexes is also important for thecellular uptake process. After binding to the target cells theDNA-polycation complex should be taken up by endocytosis. Since theendocytic vesicles have a homogenous internal diameter of about 100 nmin hepatocytes and are of similar size in other cell types, DNAcomplexes smaller than 100 nm are preferred.

Condensation of DNA

A significant number of multivalent cations with widely differentmolecular structures have been shown to induce condensation of DNA.

Two approaches for compacting (used herein as an equivalent to the termcondensing) DNA:

1. Multivalent cations with a charge of three or higher have been shownto condense DNA. These include spermidine, spermine, Co(NH₃)₆ ³⁺,Fe³⁺,and natural or synthetic polymers such as histone Hi, protamine,polylysine, and polyethylenimine. Analysis has shown DNA condensation tobe favored when 90% or more of the charges along the sugar-phosphatebackbone are neutralized. 2. Polymers (neutral or anionic) which canincrease repulsion between DNA and its surroundings have been shown tocompact DNA. Most significantly, spontaneous DNA self-assembly andaggregation process have been shown to result from the confinement oflarge amounts of DNA, due to excluded volume effect.

Depending upon the concentration of DNA, condensation leads to threemain types of structures:

1) In extremely dilute solution (about 1 μg/mL or below), long DNAmolecules can undergo a monomolecular collapse and form structuresdescribed as toroid. 2) In very dilute solution (about 10 μg/mL)microaggregates form with short or long molecules and remain insuspension. Toroids, rods and small aggregates can be seen in suchsolution. 3) In dilute solution (about 1 mg/mL) large aggregates areformed that sediment readily.

Toroids have been considered an attractive form for gene deliverybecause they have the smallest size. While the size of DNA toroidsproduced within single preparations has been shown to vary considerably,toroid size is unaffected by the length of DNA being condensed. DNAmolecules from 400 bp to genomic length produce toroids similar in size.Therefore one toroid can include from one to several DNA molecules. Thekinetics of DNA collapse by polycations that resulted in toroids is veryslow. For example, DNA condensation by Co(NH₃)₆Cl₃ needs 2 hours at roomtemperature.

The mechanism of DNA condensation is not clear. The electrostatic forcebetween unperturbed helices arises primarily from a counterionfluctuation mechanism requiring multivalent cations and plays a majorrole in DNA condensation. The hydration forces predominate overelectrostatic forces when the DNA helices approach closer then a fewwater diameters. In a case of DNA—polymeric polycation interactions, DNAcondensation is a more complicated process than the case of lowmolecular weight polycations. Different polycationic proteins cangenerate toroid and rod formation with different size DNA at a ratio ofpositive to negative charge of 0.4. T4 DNA complexes with polyarginineor histone can form two types of structures; an elongated structure witha long axis length of about 350 nm (like free DNA) and dense sphericalparticles. Both forms exist simultaneously in the same solution. Thereason for the co-existence of the two forms can be explained as anuneven distribution of the polycation chains among the DNA molecules.The uneven distribution generates two thermodynamically favorableconformations.

The electrophoretic mobility of DNA -polycation complexes can changefrom negative to positive in excess of polycation. It is likely thatlarge polycations don't completely align along DNA but form polymerloops that interact with other DNA molecules. The rapid aggregation andstrong intermolecular forces between different DNA molecules may preventthe slow adjustment between helices needed to form tightly packedorderly particles.

Cationic molecules with charge greater than +2 are able to condense DNAinto compact structures (Bloomfield V. A., DNA condensation, (1996)Curr, Opinion in Struct. Biol., 6:334-341). This phenomenon plays a rolein chromatin and viral assembly and is of particular importance in theconstruction of artificial gene delivery vectors. Morphologies ofcondensed DNA during titration of DNA with polycations are now welldocumented. When DNA is in excess (DNA/polycation charge ratio >1),complexes assemble into “daisy-shaped” particles that stabilized withloops of uncondensed DNA (Hansma, G. H., Golan, R., Hsieh, W., Lollo, C.P., Mullen-Ley, P. and Kwoh. D. (1998) DNA condensation for gene therapyas monitored by atomic force microscopy, Nucleic Acids Res.26:2481-2487). When polycation is in excess (DNA/polycation ratio <1),DNA condenses completely within particles that adopt customarily toroidmorphology (Tang, M. X., and Szoka, F. C., Jr. 1997, The influence ofpolymer structure on the interactions of cationic polymers with DNA andmorphology of the resulting complexes, Gene Ther. 4:823-832). In lowsalt aqueous solutions the excess of polycation stabilizes these highlycondensed structures and maintains them in soluble state (Kabanov A V,Kabanov V A., Interpolyelectrolyte and block ionomer complexes for genedelivery: physico-chemical aspects, Adv. Drug Delivery Rev. 30:49-60(1998)).

Several methods can be used to determine the condensation state of DNA.They include the prevention of fluorescent molecules such as ethidiumbromide from intercalating into the DNA. The condensation state of DNAwas monitored as previously described (Dash, RR, Toncheva V, Schacht E,Seymour L W J. Controlled Release 48:269-276). Alternatively thecondensation of fluorescein-labeled DNA (or any fluorescent group)causes self-quenching by bringing the fluorescent groups on the DNAcloser together (Trubetskoy, V S, Budker, V G, Slattum, P M, Hagstrom, JE and Wolff, J A. Analytical Biochemistry 267:309-313, 1999).

Preparation of Negatively-Charged (anionic) Particles

As previously stated, preparation of polycation-condensed DNA particlesis of particular importance for gene therapy, more specifically,particle delivery such as the design of non-viral gene transfer vectors.Optimal transfection activity in vitro and in vivo can require an excessof polycation molecules. However, the presence of a large excess ofpolycations may be toxic to cells and tissues. Moreover, thenon-specific binding of cationic particles to all cells forestallscellular targeting. Positive charge also has an adverse influence onbiodistribution of the complexes in vivo.

SUMMARY OF THE INVENTION

In order to avoid unwanted effects, anionic particles containing anexcess of DNA and cell receptor ligands for targeting have beendeveloped. The present invention describes a process for negativelycharging DNA particles by recharging fully condensed polycation/DNAcomplexes with polyions.

In a preferred embodiment, a process is described for delivering acomplex to a cell, comprising, forming a compound having a net chargecomprising a polyion and a polymer in a solution, adding a chargedpolymer to the solution in sufficient amount to form the complex havinga net charge different from the compound net charge; and, inserting thecomplex into a mammal.

In another preferred embodiment, a complex for delivering a polyion to acell is described, comprising a polyion and a charged polymer whereinthe polyion and the charged polymer are bound in complex, the complexhaving a net charge that is the same as the net charge of the chargedpolymer.

In another preferred embodiment a drug for delivery to a cell, isdescribed, comprising a polycation non-covalently attached to apolyanion complexed with a negatively charged polyion.

Reference is now made in detail to the preferred embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A) illustrates F1-DNA decondensation during titration ofF1-DNA/PLL complex (1:3 charge ratio, (F1-DNA)=20 μg/ml, 25 mM HEPES, pH7.5) with different polyanions; (B) titration of DNA/PLL (1:3 chargeratio, (DNA)=20 μg/ml, 25 mM HEPES, pH 7.5) complex with SPLL asassessed by light scattering methods. Intensity of scattered light (190)was measured using spectrofluorimeter. Percentage of particles <100 nmin diameter was measured using particle size analyzer as described inthe specification. COOH/NH₂ ratios were calculated on the basis of molweights of N-succinyl lysine and lysine monomers in SPLL and PLLrespectively; (C) potential changes during titration of DNA/PLL complex(1:3 charge ratio, (DNA)=20 μg/ml, 25 mM HEPES, pH 7.5) with SPLL.

FIG. 2 illustrates AFM images of DNA/PLL/SPLL complexes (1:3:10 initialratio) absorbed on mica in 25 mM HEPES, pH 7.5 as described in thespecification.

FIG. 3(A) illustrates visible spectra of DNA complexes isolated afterRh-DNA/F1-PLL/SPLL (1:3:10) ultracentrifugation and Rh-DNA/F1-PLL (1:1)standard dissolved in 2.5 M NaCl; (B) visible spectra of DNA complexesisolated after Rh-DNA/PLL/F1-SPLL (1:3:10) ultracentrifugation andRh-DNA/F1-SPLL (1:1) standard in the same conditions. FIG. 4 illustratestransfection efficacy of DNA/PEI complexes recharged with increasingamounts of SPLL polyanion. DNA/PEI/SPLL complexes (2 μg DNA, 4 μg PEI)were added to HUH7 cells in bovine serum. After 4 hrs of incubationserum with DNA was replaced with fresh OPTI-MEM culture medium with 10%fetal serum. Cells were harvested for luciferase assay 48 hrs aftertransfection.

DETAILED DESCRIPTION

Abbreviations: Poly-L-Lysine (PLL), succinic anhydride-PLL (SPLL),polymethacrylic acid, pMAA and polyaspartic acid, pAsp

Gene therapy research may involve the biological pH gradient that isactive within organisms as a factor in delivering a polynucleotide to acell. Different pathways that may be affected by the pH gradient includecellular transport mechanisms, endosomal disruption/breakdown, andparticle disassembly (release of the DNA).

Gradients that can be useful in gene therapy research involve ionicgradients that are related to cells. For example, both Na+ and K+ havelarge concentration gradients that exist across the cell membrane.Recharging systems can utilize such gradients to influence delivery of apolynucleotide to a cell. DNA can be compacted by adding polycations tothe mixture. By interacting an appropriate cation with a DNA containingsystem, DNA condensation can take place. Since the ion utilized forcompaction may exist in higher concentration outside of the cellmembrane compared to inside the cell membrane, this natural ionicgradient can be utilized in delivery systems.

Polymers

A polymer is a molecule built up by repetitive bonding together ofsmaller units called monomers. In this application the term polymerincludes both oligomers which have two to about 80 monomers and polymershaving more than 80 monomers. The polymer can be linear, branchednetwork, star, comb, or ladder types of polymer. The polymer can be ahomopolymer in which a single monomer is used or can be copolymer inwhich two or more monomers are used. Types of copolymers includealternating, random, block and graft.

To those skilled in the art of polymerization, there are severalcategories of polymerization processes that can be utilized in thedescribed process. The polymerization can be chain or step. Thisclassification description is more often used that the previousterminology of addition and condensation polymer.

Step Polymerization: In step polymerization, the polymerization occursin a stepwise fashion. Polymer growth occurs by reaction betweenmonomers, oligomers and polymers. No initiator is needed since there isthe same reaction throughout and there is no termination step so thatthe end groups are still reactive. The polymerization rate decreases asthe functional groups are consumed.

Typically, step polymerization is done either of two different ways. Oneway, the monomer has both reactive functional groups (A and B) in thesame molecule so that

A-B yields -[A-B]-

Or the other approach is to have two difunctional monomers.

A-A+B-B yields -[A-A-B-B]-

Generally, these reactions can involve acylation or alkylation.Acylation is defined as the introduction of an acyl group (—COR) onto amolecule. Alkylation is defined as the introduction of an alkyl grouponto a molecule.

“If functional group A is an amine then B can be (but not restricted to)an isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide,sulfonyl chloride, aldehyde (including formaldehyde and glutaraldehyde),ketone, epoxide, carbonate, imidoester, carboxylate activated with acarbodiimide, alkylphosphate, arylhalides (difluoro-dinitrobenzene),anhydride, or acid halide, p-nitrophenyl ester, o-nitrophenyl ester,pentachlorophenyl ester, pentafluorophenyl ester, carbonylimidazole,carbonyl pyridinium, or carbonyl dimethylaminopyridinium. In other termswhen function A is an amine then function B can be acylating oralkylating agent or amination agent.

If functional group A is a sulfhydryl then function B can be (but notrestricted to) an iodoacetyl derivative, maleimide, aziridinederivative, acryloyl derivative, fluorobenzene derivatives, or disulfidederivative (such as a pyridyl disulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives).

If functional group A is carboxylate then function B can be (but notrestricted to) adiazoacetate or an amine in which a carbodiimide isused. Other additives may be utilized such as carbonyldiimidazole,dimethylamino pyridine (DMAP), N-hydroxysuccinimide or alcohol usingcarbodiimide and DMAP.

If functional group A is an hydroxyl then function B can be (but notrestricted to) an epoxide, oxirane, or an amine in whichcarbonyldiimidazole or N, N′-disuccinimidyl carbonate, orN-hydroxysuccinimidyl chloroformate or other chloroformates are used. Iffunctional group A is an aldehyde or ketone then function B can be (butnot restricted to) an hydrazine, hydrazide derivative, amine (to form aSchiff Base that may or may not be reduced by reducing agents such asNaCNBH₃ ) or hydroxyl compound to form a ketal or acetal.

Yet another approach is to have one bifunctional monomer so that A-Aplus another agent yields -[A-A]-. If function A is a sulfhydryl groupthen it can be converted to disulfide bonds by oxidizing agents such asiodine (I₂) or NaIO₄ (sodium periodate), or oxygen (O₂). Function A canalso be an amine that is converted to a sulfhydryl group by reactionwith 2-Iminothiolate (Traut's reagent) which then undergoes oxidationand disulfide formation. Disulfide derivatives (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid {TNB} derivatives) can also beused to catalyze disulfide bond formation. Functional group A or B inany of the above examples could also be a photoreactive group such asaryl azide (including halogenated aryl azide), diazo, benzophenone,alkyne or diazirine derivative.

Reactions of the amine, hydroxyl, sulfhydryl, carboxylate groups yieldchemical bonds that are described as amide, amidine, disulfide, ethers,esters, enamine, imine, urea, isothiourea, isourea, sulfonamide,carbamate, alkylamine bond (secondary amine), carbon-nitrogen singlebonds in which the carbon contains a hydroxyl group, thioether, diol,hydrazone, diazo, or sulfone.

If functional group A is an aldehyde or ketone then function B can be(but not restricted to) an hydrazine, hydrazide derivative, amine (toform a Schiff Base that may or may not be reduced by reducing agentssuch as NaCNBH₃) or hydroxyl compound to form a ketal or acetal.

Yet another approach is to have one difunctional monomer so that

A-A plus another agent yields -[A-A]-.

If function A is a sulfhydryl group then it can be converted todisulfide bonds by oxidizing agents such as iodine (I₂) or NaIO₄ (sodiumperiodate), or oxygen (O₂). Function A can also be an amine that isconverted to a sulfhydryl group by reaction with 2-iminothiolate(Traut's reagent) which then undergoes oxidation and disulfideformation. Disulfide derivatives (such as a pyridyl disulfide or5-thio-2-nitrobenzoic acid {TNB} derivatives) can also be used tocatalyze disulfide bond formation.

Functional group A or B in any of the above examples could also be aphotoreactive group such as aryl azides, halogenated aryl azides, diazo,benzophenones, alkynes or diazirine derivatives.

Reactions of the amine, hydroxyl, sulfhydryl, carboxylate groups yieldchemical bonds that are described as amide, amidine, disulfide, ethers,esters, enamine, urea, isothiourea, isourea, sulfonamide, carbamate,carbon-nitrogen double bond (imine), alkylamine bond (secondary amine),carbon-nitrogen single bonds in which the carbon contains a hydroxylgroup, thio- ether, diol, hydrazone, diazo, or sulfone.

Chain Polymerization: In chain-reaction polymerization growth of thepolymer occurs by successive addition of monomer units to limited numberof growing chains. The initiation and propagation mechanisms aredifferent and there is usually a chain-terminating step. Thepolymerization rate remains constant until the monomer is depleted.

Monomers containing vinyl, acrylate, methacrylate, acrylamide,methacrylamide groups can undergo chain reaction which can be radical,anionic, or cationic. Chain polymerization can also be accomplished bycycle or ring opening polymerization. Several different types of freeradical initiators could be used that include peroxides, hydroxyperoxides, and azo compounds such as 2,2′-Azobis(-amidinopropane)dihydrochloride (AAP). A compound is a material made up of two or moreelements.

Types of Monomers: A wide variety of monomers can be used in thepolymerization processes. These include positive charged organicmonomers such as amines, imidine, guanidine, imine, hydroxylamine,hydrozyine, and heterocycles (like imidazole, pyridine, morpholine,pyrimidine, or pyrene). The amines could be pH-sensitive in that the pKaof the amine is within the physiologic range of 4-8. Specific aminesinclude spermine, spermidine, N,N′-bis(2-aminoethyl)-1,3-propanediamine(AEPD), and 3,3′-Diamino-N,N-dimethyldipropylammonium bromide.

Monomers can also be hydrophobic, hydrophilic or amphipathic.Amphipathic compounds have both hydrophilic (water-soluble) andhydrophobic (water-insoluble) parts. Hydrophilic groups indicate inqualitative terms that the chemical moiety is water-preferring.Typically, such chemical groups are water soluble, and are hydrogen bonddonors or acceptors with water. Examples of hydrophilic groups includecompounds with the following chemical moieties carbohydrates;polyoxyethylene, peptides, oligonucleotides and groups containingamines, amides, alkoxy amides, carboxylic acids, sulfurs, or hydroxyls.Hydrophobic groups indicate in qualitative terms that the chemicalmoiety is water-avoiding. Typically, such chemical groups are not watersoluble, and tend not to hydrogen bond. Hydrocarbons are hydrophobicgroups. Monomers can also be intercalating agents such as acridine,thiazole orange, or ethidium bromide.

Other Components of the Monomers and Polymers: The polymers have othergroups that increase their utility. These groups can be incorporatedinto monomers prior to polymer formation or attached to the polymerafter its formation. These groups include: Targeting Groups- such groupsare used for targeting the polymer-nucleic acid complexes to specificcells or tissues. Examples of such targeting agents include agents thattarget to the asialoglycoprotein receptor by using asialoglycoproteinsor galactose residues. Other proteins such as insulin, EGF, ortransferrin can be used for targeting. Protein refers to a molecule madeup of 2 or more amino acid residues connected one to another as in apolypeptide. The amino acids may be naturally occurring or synthetic.Peptides that include the RGD sequence can be used to target many cells.Peptide refers to a linear series of amino acid residues connected toone another by peptide bonds between the alpha-amino group and carboxylgroup of contiguous amino acid residues. Chemical groups that react withsulfhydryl or disulfide groups on cells can also be used to target manytypes of cells. Folate and other vitamins can also be used fortargeting. Other targeting groups include molecules that interact withmembranes such as fatty acids, cholesterol, dansyl compounds, andamphotericin derivatives.

After interaction of the supramolecular complexes with the cell, othertargeting groups can be used to increase the delivery of the drug ornucleic acid to certain parts of the cell. For example, agents can beused to disrupt endosomes and a nuclear localizing signal (NLS) can beused to target the nucleus.

A variety of ligands have been used to target drugs and genes to cellsand to specific cellular receptors. The ligand may seek a target withinthe cell membrane, on the cell membrane or near a cell. Binding ofligands to receptors typically initiates endocytosis. Ligands that bindto receptors that are not endocytosed could also be used for DNAdelivery. For example, peptides containing RGD peptide sequence thatbind integrin receptor could be used. In addition viral proteins couldbe used to bind the complex to cells. Lipids and steroids could be usedto directly insert a complex into cellular membranes.

The polymers can also contain cleavable groups within themselves. Whenattached to the targeting group, cleavage leads to reduce interactionbetween the complex and the receptor for the targeting group. Cleavablegroups include but are not restricted to disulfide bonds, diols, diazobonds, ester bonds, sulfone bonds, acetals, ketals, enol ethers, enolesters, enamines and imines.

Reporter or marker molecules are compounds that can be easily detected.Typically they are fluorescent compounds such as fluorescein, rhodamine,Texas red, CY-5, CY-3 or dansyl compounds. They can be molecules thatcan be detected by UV or visible spectroscopy or by antibodyinteractions or by electron spin resonance. Biotin is another reportermolecule that can be detected by labeled avidin. Biotin could also beused to attach targeting groups.

A polycation is a polymer containing a net positive charge, for examplepoly-L-lysine hydrobromide. The polycation can contain monomer unitsthat are charge positive, charge neutral, or charge negative, however,the net charge of the polymer must be positive. A polycation also canmean a non-polymeric molecule that contains two or more positivecharges. A polyanion is a polymer containing a net negative charge, forexample polyglutamic acid. The polyanion can contain monomer units thatare charge negative, charge neutral, or charge positive, however, thenet charge on the polymer must be negative. A polyanion can also mean anon-polymeric molecule that contains two or more negative charges. Theterm polyion includes polycation, polyanion, zwitterionic polymers, andneutral polymers. The term zwitterionic refers to the product (salt) ofthe reaction between an acidic group and a basic group that are part ofthe same molecule. Salts are ionic compounds that dissociate intocations and anions when dissolved in solution. Salts increase the ionicstrength of a solution, and consequently decrease interactions betweennucleic acids with other cations. A charged polymer is a polymer thatcontains residues, monomers, groups, or parts with a positive ornegative charge and whose net charge can be neutral, positive, ornegative.

Signals

In a preferred embodiment, a chemical reaction can be used to attach asignal to a nucleic acid complex. The signal is defined in thisspecification as a molecule that modifies the nucleic acid complex andcan direct it to a cell location (such as tissue cells) or location in acell (such as the nucleus) either in culture or in a whole organism. Bymodifying the cellular or tissue location of the foreign gene, theexpression of the foreign gene can be enhanced.

The signal can be a protein, peptide, lipid, steroid, sugar,carbohydrate, nucleic acid or synthetic compound. The signals enhancecellular binding to receptors, cytoplasmic transport to the nucleus andnuclear entry or release from endosomes or other intracellular vesicles.

Nuclear localizing signals enhance the targeting of the gene intoproximity of the nucleus and/or its entry into the nucleus. Such nucleartransport signals can be a protein or a peptide such as the SV40 large Tag NLS or the nucleoplasmin NLS. These nuclear localizing signalsinteract with a variety of nuclear transport factors such as the NLSreceptor (karyopherin alpha) which then interacts with karyopherin beta.The nuclear transport proteins themselves could also function as NLS'ssince they are targeted to the nuclear pore and nucleus.

Signals that enhance release from intracellular compartments (releasingsignals) can cause DNA release from intracellular compartments such asendosomes (early and late), lysosomes, phagosomes, vesicle, endoplasmicreticulum, Golgi apparatus, trans Golgi network (TGN), and sarcoplasmicreticulum. Release includes movement out of an intracellular compartmentinto cytoplasm or into an organelle such as the nucleus. Releasingsignals include chemicals such as chloroquine, bafilomycin or BrefeldinA1 and the ER-retaining signal (KDEL sequence), viral components such asinfluenza virus hemagglutinin subunit HA-2 peptides and other types ofamphipathic peptides.

Cellular receptor signals are any signal that enhances the associationof the gene or particle with a cell. This can be accomplished by eitherincreasing the binding of the gene to the cell surface and/or itsassociation with an intracellular compartment, for example: ligands thatenhance endocytosis by enhancing binding the cell surface. This includesagents that target to the asialoglycoprotein receptor by usingasialoglycoproteins or galactose residues. Other proteins such asinsulin, EGF, or transferrin can be used for targeting. Peptides thatinclude the RGD sequence can be used to target many cells. Chemicalgroups that react with sulfhydryl or disulfide groups on cells can alsobe used to target many types of cells. Folate and other vitamins canalso be used for targeting. Other targeting groups include moleculesthat interact with membranes such as lipids fatty acids, cholesterol,dansyl compounds, and amphotericin derivatives. In addition viralproteins could be used to bind cells.

The present invention provides compounds used in systems for thetransfer of polynucleotides, oligonucleotides, and other compounds intoassociation with cells within tissues in situ and in vivo.

The process of delivering a polynucleotide to a cell has been commonlytermed “transfection” or the process of “transfecting” and also it hasbeen termed “transformation”. The polynucleotide could be used toproduce a change in a cell that can be therapeutic. The delivery ofpolynucleotides or genetic material for therapeutic and researchpurposes is commonly called “gene therapy”. The polynucleotides orgenetic material being delivered are generally mixed with transfectionreagents prior to delivery.

A biologically active compound is a compound having the potential toreact with biological components. More particularly, biologically activecompounds utilized in this specification are designed to change thenatural processes associated with a living cell. For purposes of thisspecification, a cellular natural process is a process that isassociated with a cell before delivery of a biologically activecompound. In this specification, the cellular production of, orinhibition of a material, such as a protein, caused by a human assistinga molecule to an in vivo cell is an example of a delivered biologicallyactive compound. Pharmaceuticals, proteins, peptides, polypeptides,hormones, cytokines, antigens, viruses, oligonucleotides, and nucleicacids are examples of biologically active compounds.

The term “nucleic acid” is a term of art that refers to a polymercontaining at least two nucleotides. “Nucleotides” contain a sugardeoxyribose (DNA) or ribose (RNA), a base, and a phosphate group.Nucleotides are linked together through the phosphate groups. “Bases”include purines and pyrimidines, which further include natural compoundsadenine, thymine, guanine, cytosine, uracil, inosine, and syntheticderivatives of purines and pyrimidines, or natural analogs. Nucleotidesare the monomeric units of nucleic acid polymers. A “polynucleotide” isdistinguished here from an “oligonucleotide” by containing more than 80monomeric units; oligonucleotides contain from 2 to 80 nucleotides. Theterm nucleic acid includes deoxyribonucleic acid (DNA) and ribonucleicacid (RNA). DNA may be in the form of anti-sense, plasmid DNA, parts ofa plasmid DNA, vectors (P1, PAC, BAC, YAC, and artificial chromosomes),expression cassettes, chimeric sequences, chromosomal DNA, orderivatives of these groups. RNA may be in the form of oligonucleotideRNA, tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomalRNA), mRNA (messenger RNA), anti-sense RNA, ribozymes, chimericsequences, or derivatives of these groups. “Anti-sense” is apolynucleotide that interferes with the function of DNA and/or RNA. Thismay result in suppression of expression. Natural nucleic acids have aphosphate backbone, artificial nucleic acids may contain other types ofbackbones, nucleotides, or bases. These include PNAs (peptide nucleicacids), phosphothionates, and other variants of the phosphate backboneof native nucleic acids. In addition, DNA and RNA may be single, double,triple, or quadruple stranded. “Expression cassette” refers to a naturalor recombinantly produced polynucleotide molecule which is capable ofexpressing protein(s). A DNA expression cassette typically includes apromoter (allowing transcription initiation), and a sequence encodingone or more proteins. Optionally, the expression cassette may includetranscriptional enhancers, non-coding sequences, splicing signals,transcription termination signals, and polyadenylation signals. An RNAexpression cassette typically includes a translation initiation codon(allowing translation initiation), and a sequence encoding one or moreproteins. Optionally, the expression cassette may include translationtermination signals, a polyadenosine sequence, internal ribosome entrysites (IRES), and non-coding sequences.

The term “naked polynucleotides” indicates that the polynucleotides arenot associated with a transfection reagent or other delivery vehiclethat is required for the polynucleotide to be delivered to the cardiacmuscle cell. A “transfection reagent” is a compound or compounds used inthe prior art that bind(s) to or complex(es) with oligonucleotides andpolynucleotides, and mediates their entry into cells. The transfectionreagent also mediates the binding and internalization ofoligonucleotides and polynucleotides into cells. Examples oftransfection reagents include cationic liposomes and lipids, polyamines,calcium phosphate precipitates, histone proteins, polyethylenimine, andpolylysine complexes. It has been shown that cationic proteins likehistones and protamines, or synthetic polymers like polylysine,polyarginine, polyornithine, DEAE dextran, polybrene, andpolyethylenimine may be effective intracellular delivery agents, whilesmall polycations like spermine may be ineffective. Typically, thetransfection reagent has a net positive charge that binds to theoligonucleotide's or polynucleotide's negative charge. The transfectionreagent mediates binding of oligonucleotides and polynucleotides tocells via its positive charge (that binds to the cell membrane'snegative charge) or via ligands that bind to receptors in the cell. Forexample, cationic liposomes or polylysine complexes have net positivecharges that enable them to bind to DNA or RNA. Polyethylenimine, whichfacilitates gene expression without additional treatments, probablydisrupts endosomal function itself.

Other vehicles are also used, in the prior art, to transfer genes intocells. These include complexing the polynucleotides on particles thatare then accelerated into the cell. This is termed “biolistic” or “gun”techniques. Other methods include “electroporation,” in which a deviceis used to give an electric charge to cells. The charge increases thepermeability of the cell.

Ionic (electrostatic) interactions are the non-covalent association oftwo or more substances due to attractive forces between positive andnegative charges, or partial positive and partial negative charges.

Condensed Nucleic Acids: Condensing a polymer means decreasing thevolume that the polymer occupies. An example of condensing nucleic acidis the condensation of DNA that occurs in cells. The DNA from a humancell is approximately one meter in length but is condensed to fit in acell nucleus that has a diameter of approximately 10 microns. The cellscondense (or compacts) DNA by a series of packaging mechanisms involvingthe histones and other chromosomal proteins to form nucleosomes andchromatin. The DNA within these structures is rendered partiallyresistant to nuclease DNase) action. The process of condensing polymerscan be used for delivering them into cells of an organism.

A delivered polymer can stay within the cytoplasm or nucleus apart fromthe endogenous genetic material. Alternatively, the polymer couldrecombine (become a part of) the endogenous genetic material. Forexample, DNA can insert into chromosomal DNA by either homologous ornon-homologous recombination.

Condensed nucleic acids may be delivered intravasculary,intra-arterially, intravenously, orally, intraduodenaly, via the jejunum(or ileum or colon), rectally, transdermally, subcutaneously,intramuscularly, intraperitoneally, intraparenterally, via directinjections into tissues such as the liver, lung, heart, muscle, spleen,pancreas, brain (including intraventricular), spinal cord, ganglion,lymph nodes, lymphatic system, adipose tissues, thyroid tissue, adrenalglands, kidneys, prostate, blood cells, bone marrow cells, cancer cells,tumors, eye retina, via the bile duct, or via mucosal membranes such asin the mouth, nose, throat, vagina or rectum or into ducts of thesalivary or other exocrine glands. “Delivered” means that thepolynucleotide becomes associated with the cell. The polynucleotide canbe on the membrane of the cell or inside the cytoplasm, nucleus, orother organelle of the cell.

An intravascular route of administration enables a polymer orpolynucleotide to be delivered to cells more evenly distributed and moreefficiently expressed than direct injections. Intravascular herein meanswithin a tubular structure called a vessel that is connected to a tissueor organ within the body. Within the cavity of the tubular structure, abodily fluid flows to or from the body part. Examples of bodily fluidinclude blood, lymphatic fluid, or bile. Examples of vessels includearteries, arterioles, capillaries, venules, sinusoids, veins,lymphatics, and bile ducts. The intravascular route includes deliverythrough the blood vessels such as an artery or a vein.

An administration route involving the mucosal membranes is meant toinclude nasal, bronchial, inhalation into the lungs, or via the eyes.

Recharging Condensed Nucleic Acids

Polyions for gene therapy and gene therapy research can involve anionicsystems as well as charge neutral or charge-positive systems. The ionicpolymer can be utilized in “recharging” (another layer having adifferent charge) the condensed polynucleotide complex. The resultingrecharged complex can be formed with an appropriate amount of chargesuch that the resulting complex has a net negative, positive or neutralcharge. The interaction between the polycation and the polyanion can beionic, can involve the ionic interaction of the two polymer layers withshared cations, or can be crosslinked between cationic and anionic siteswith a crosslinking system (including cleavable crosslinking systems,such as those containing disulfide bonds). The interaction between thecharges located on the two polymer layers can be influenced with the useof added ions to the system. With the appropriate choice of ion, thelayers can be made to disassociate from one another as the ion diffusesfrom the complex into the cell in which the concentration of the ion islow (use of an ion gradient).

Electrostatic complexes between water-soluble polyelectrolytes have beenstudied widely in recent ears. Complexes containing DNA as a polyanionicconstituent only recently came to the attention because of theirpotential use in gene therapy applications such as non-viral genetransfer preparations (polyplexes) for particle delivery to a cell.Strong polyelectrolytes, polyanion/polycation complexes, are usuallyformed at a 1:1 charge stoichiometrically. A charge ratio 1:1 complexbetween DNA and Poly-L-Lysine (PLL) also has been demonstrated in theprior art.

Polyanions effectively enhance the gene delivery/gene expressioncapabilities of all major classes of polycation gene delivery reagents.In that regard, we disclose the formation of negatively charged tertiarycomplexes containing nucleic acid, PLL, and succinic anhydride-PLL(SPLL) complexes. SPLL is added to a cationic nucleic acid/PLL complexin solution. Nucleic acid at the core of such complexes remainscondensed, in the form of particles ˜50 nm in diameter. DNA and PLLbinds SPLL in 1:1:1 complex with SPLL providing a net negative charge tothe entire complex. Such small negatively charged particles are usefulfor non-viral gene transfer applications.

One of the advantages that flow from recharging DNA particles isreducing their non-specific interactions with cells and serum proteins[(Wolfert et al. Hum. Gene Therapy 7:2123-2133 (1996); Dash et al., GeneTherapy 6:643-650 (1999); Plank et al., Hum. Gene Ther. 7:1437-1446(1996); Ogris et al., Gene Therapy 6:595-605 (1999); Schacht et al.Brit. Patent Application 9623051.1 (1996)]

A wide a variety of polyanions can be used to recharge theDNA/polycation particles. They include (but not restricted to): Anywater-soluble polyanion can be used for recharging purposes includingsuccinylated PLL, succinylated PEI (branched), polyglutamic acid,polyaspartic acid, polyacrylic acid, polymethacrylic acid,polyethylacrylic acid, polypropylacrylic acid, polybutylacrylic acid,polymaleic acid, dextran sulfate, heparin, hyaluronic acid,polysulfates, polysulfonates, polyvinyl phosphoric acid, polyvinylphosphonic acid, copolymers of polymaleic acid, polyhydroxybutyric acid,acidic polycarbohydrates, DNA, RNA, negatively charged proteins,pegylated derivatives of above polyanions, pegylated derivativescarrying specific ligands, block and graft copolymers of polyanions andany hydrophilic polymers (PEG, poly(vinylpyrrolidone), poly(acrylamide),etc).

These polyanions can be added prior to the nucleic acid complex beingdelivered to the cell or organism. In one preferred embodiment therecharged nucleic acid complexes (polyanion/polycation/nucleic acidcomplex) are formed in a container and then administered to the cell ororganism. In another preferred embodiment, the polycation/nucleic acidcomplex is recharged with a polyion prior to delivery to the organismand the nucleic acid remains condensed. In this embodiment the nucleicacid can remain more than 50%, 60%, 70%, 80%, 90% or 100% condensed aswell.

When an excess of polyion is present, DNA forms soluble condensed(toroid) structures stabilized with an excess of polyion. When, inaddition to this binary complex, a third polyelectrolyte is present, atertiary complex exists. In the absence of salt such tertiary complexmight exist indefinitely. If the last added polyion is in excess, itstabilizes the complex in the form of a soluble colloid. Using thismethod, a DNA/polycation complex, which maintains a net positive charge,reverses its charge and becomes “recharged”. The complex can be designed(e.g. choice of polycation and polyanion, presence of crosslinking) sothat in the presence of salt, the complex dissociates into binarycomplex and free excess of third polyion.

In general, tertiary DNA/PLL/SPLL complex exhibit the same colloidproperties as binary DNA/PLL complex. In low salt solution it formsflocculate around PLL/SPLL charge equivalence point (FIG. 1).

DNA condensation assays based on the effect of concentration-dependentself-quenching of covalently-bound fluorophores upon DNA collapseindicated essentially the same phenomenon described in the prior art.Polyanions with high charge density (polymethacrylic acid, pMAA andpolyaspartic acid, pAsp) were able to decondense DNA prior to those thatcomplexed with PLL while polyanions with lower charge density(polyglutamic acid, pGlu, SPLL) failed to decondense DNA (FIG. 1).Together with z-potential measurements (FIG. 3), these data representsupport for the presence of negatively charged condensed DNA particles.These particles are approximately 50 nm in diameter in low salt bufferas measured by atomic force microscopy (FIG. 2) which revealed particlesof spheroid morphology. This places them very close in size to binaryDNA/PLL particles.

The issue of stoichiometry in such tertiary complexes is of primaryimportance to determine how much polyanion is associated with DNA afterformation of tertiary complex and potential dissociation of polycationafter polyanion binding. We developed a methodology for DNA complexstoichiometry determination which includes step density gradientultracentrifugation of complexes prepared with fluorescently labeledDNA, PLL and SPLL. Retrieved complexes were always found aggregated andpossess DNA/PLL/SPLL (1:1:1) stoichiometry. This surprising findingassumes major redistribution of charges inside the particle since netcharge of the complex is negative. Excess PLL was found to complex withany excess SPLL.

In another preferred embodiment, the polyanion can be covalentlyattached to the polycation using a variety of chemical reactions withoutthe use of crosslinker. The polyanion can contain reactive groups thatcovalently attach to groups on the polycation. The types of reactionsare similar to those discussed above in the section on steppolymerization.

In another preferred embodiment the attachment of the recharged complexcan be enhanced by using chelators and crown ethers, preferablypolymeric.

Excess of the polycations or polyanions can be toxic or interfere withnucleic acid delivery and transfection. In one preferred embodiment theDNA/polycation complexes are initially formed by adding only a smallexcess of polycation to nucleic acid (in charge ratio which is definedas ratio of polycation total charge to polyanion total charge at givenpH). The charge ratio of polycation to nucleic acid charge could be lessthan 2, less than 1.7, less than 1.5 or even less than 1.3. This wouldbe preferably done in low ionic strength solution so as to avoid thecomplexes from flocculation. Low ionic strength solution means solutionwith total monovalent salt concentration less than 50 mM. Then thepolyanion is added to the mixture and only a small amount of “blank”particles are formed. “Blank” particles are particles that contain onlypolycation and polyanion and no nucleic acid.

In another preferred embodiment, the polycation is added to the nucleicacid in charge excess but the excess polycation that is not in complexwith the nuclei acid is removed by purification. Purification meansremoving of charged polymer using centrifugation, dialysis,chromatography, electrophoresis, precipitation, extraction.

Yet in another preferred embodiment an ultracentrifugation procedure(termed “centrifugation step”) is used to reduce the amount of excesspolycation, polyanion, or “blank” particles. The method is based on thephenomenon that only dense DNA-containing particles can be centrifugedthrough 10% sucrose solution at 25,000 g. After centrifugation purifiedcomplex is at the bottom of the tube while excess of polyanion and“blank” particles stay on top. In modification of this experiment 40%solution of metrizamide can be used as a cushion to collect purifiedDNA/polycation/polyanion complex on the boundary for easy retrieval.

The attachment of the polyanion to the DNA/polycation complex enhancestability but can also enable a ligand or signal to be attached to theDNA particle. This is accomplished by attaching the ligand or signal tothe polyanion which in turn is attached to the DNA particle. A dialysisstep or centrifugation step can be used to reduce the amount of freepolyanion containing a ligand or signal that is in solution and notcomplexed with the DNA particle. One approach is to replace the free,uncomplexed polyanion containing a ligand or signal with free polyanionthat does not contain a ligand or signal.

Yet in another preferred embodiment a polyanion used for charge reversalis modified with neutral hydrophilic polymer for steric stabilization ofthe whole complex. The complex formation of DNA with pegylatedpolycations results in substantial stabilization of the complexestowards salt- and serum-induced flocculation (Wolfert et al. Hum. GeneTherapy 7:2123-2133 (1996), Ogris et al., Gene Therapy 6:595-605 (1999).We have demonstrated that modification of polyanion in triple complexalso significantly enhances salt and serum stability.

In another preferred embodiment a polyanion used for charge reversal iscleavable. One can imagine two ways to design a cleavable polyion: 1. apolyion cleavable in backbone, 2. a polyion cleavable in side chain.First scenario would comprise monomers linked by labile bonds such asdisulfide, diols, diazo, ester, sulfone, acetal, ketal, enol ether, enolester, imine and enamine bonds. Second scenario would involve reactivegroups (i.e. electrophiles and nucleophiles) in close proximity so thatreaction between them is rapid. Examples include having carboxylic acidderivatives (acids, esters and amides) and alcohols, thiols, carboxylicacids or amines in the same molecule reacting together to make esters,thiol esters, anhydrides or amides. In one specific preferred embodimentthe polyion contains an ester acid such as citraconic acid, ordimethylmaleyl acid that is connected to a carboxylic, alcohol, or aminegroup on the polyion.

Cleavable means that a chemical bond between atoms is broken. Labilealso means that a chemical bond between atoms is breakable. Crosslinkingrefers to the chemical attachment of two or more molecules with abifunctional reagent. A bifunctional reagent is a molecule with tworeactive ends. The reactive ends can be identical as in ahomobifunctional molecule, or different as in a heterobifunctionalmolecule.

EXAMPLES Example 1

Materials. Plasmid DNA (pCILuc) used for the condensation studies wasprovided by Bayou Biolabs, Harahan, La. Poly-L-lysine (PLL) (MW 34 kDa),poly-L-aspartic acid (pAsp) (MW 36 kDa), poly-L-glutamic acid (PLG) (MW49 kDa) and rhodamine B isothiocyanate were products of Sigma (St.Louis, Mo.). Polymethacrylic acid (PMA), metrizamide and fluoresceineisothiocyanate were from Aldrich (Milwaukee, Wis.). LABELIT® kits (MirusCorp., Madison, Wis.) were used for covalent labeling DNA withfluorescein and rhodamine.

Synthesis of succinylated PLL (SPLL). Succinic anhydride (30 mg)dissolved in 150 μl DMSO were added to PLL (20 mg) dissolved in 1 ml of0. 1 M sodium tetraborate solution in two portions. After 10 minincubation at room temperature, the polymer was precipitated with twovolumes of isopropanol with subsequent reconstitution with deionizedwater.

Labeling of PLL and DNA with fluorescein and rhodamine. Fluoresceinisothiocyanate (0.37 mg in 5 μl DMSO) was added to PLL (20 mg) in 1 mlof sodium tetraborate and incubated for 1 hr. Resulting F1-PLL waspurified by isopropanol precipitation. F1-PLL was used also forpreparation of F1-SPLL by succinylation as described above. For DNAlabeling, DNA and LABELIT® reagent (Mirus Corp., Madison, Wis.) weremixed in HEPES buffer (25 mM HEPES, pH 7.5) in reagent/DNA weight ratiosof 1:1 and incubated for 1 hr at 37° C. Labeled DNA was precipitated twotimes with NaCl/ethanol mixture (final NaCl concentration was 0.2 M,ethanol 66%) and immediately redissolved in deionized water

DNA/polyion complex formation. DNA/PLL/SPLL complexes were formed in 25mM HEPES, pH 7.5 at DNA concentration 20-100 μg/ml. The complex withDNA/PLL charge ratio (1:3) was formed by consecutive addition of PLL andthen various amounts of SPLL and vortexing for 30 sec.

Light scattering and zeta-potential measurements. Intensity of scatteredlight measured at 90° angle (I90) was estimated using Shimadzu RF 1501set at ex=600 nm; em=600 nm. Particle sizing and zeta -potentialmeasurements were performed using a Zeta Plus Particle Analyzer(Brookhaven Instruments Corp., Holtsville, N.Y.), with a laserwavelength of 532 nm.

Atomic force microscopy. Images of DNA particles were obtained usingBioProbe AFM microscope (Park Scientific instruments, Sunnyvale,Calif.). Samples (DNA concentration 1 μg/ml in 25 mM HEPES, pH 7.5) wereallowed to adsorb on mica in the presence of 1 mM NiCl₂ for 5 min andthen were viewed in the buffer in a contact mode.

Ultracentrifugation experiments. For stoichiometry studies, tertiarycomplexes were formed using fluorescently labeled polyions. Two types ofcomplexes were formed in 25 mM HEPES, pH 7.5, (charge ratio 1:3:10): a)Rh-DNA/F1-PLL/ SPLL and b) Rh-DNA/PLL/F1-SPLL. The samples (1 ml) werelayered on top of 10% sucrose solution (10 ml) with 1 ml of 40%metrizamide cushion on the bottom and were centrifuged in SW-41 Beckmanrotor in Optima LE-80K ultracentrifuge at 30,000 rpm for 20 min.DNA-containing complexes were retrieved from sucrose/metrizamideboundary using Pasteur pipet and were dissolved in 2.5 M NaCl solution.Visible spectra of the complexes and 1:1 premixed Rh-DNA/F1-PLL andRh-DNA/F1-SPLL standards (700-400 nm) were recorded using Shimadzu UV1601 spectrophotometer.

Example 2

Recharging of Polyion Condensed DNA Particles: The chief DNA/polycationcomplex used was DNA/PLL (1:3 charge ratio) formed in low salt buffer.At these conditions, plasmid DNA is completely condensed and compactedinto toroid-shaped soluble particles stabilized with excess of polyion(Kabanov et al. Adv. Drug Delivery Rev 30:49-60 (1998). The DNAparticles were characterized after addition of a third polyion componentto such binary DNA/polyion complex. It has been shown that polyanion(polymer or negatively-charged lipid bilayer) can release DNA from itscomplex with cationic liposomes. As judged by DNA condensation assaybased on ethidium bromide binding, upon addition of such polyanions asdextran sulfate or heparin to the D NA/DOTAP lipid complexes results inrelease of free DNA. Using a fluorescein-labeled DNA condensation assay(Trubetskoy et al. Anal. Biochem. 267:309-313(1999) we demonstrate thatthe same is true for DNA/synthetic polyion complexes (FIG. 1A).

The aggregation state of condensed DNA particles was determined usingboth static and dynamic light scattering techniques. Upon titration ofDNA/PLL (1:3) complex with increasing amounts of SPLL in low saltsolution, turbidity of the reaction mixture, an indication ofaggregation, increases when the lysine to lysyl succinate (NH₂/COOH)ratio approaches 1:1 (FIG. 1(B)). With an excess of polyanion, turbiditydecreases. Correspondingly, assessment of particle size by dynamic lightscattering shows that small DNA particles (<100 nm) exist before andafter the equivalent point. Large aggregates are present only at a 1:1charge ratio of polyion to polyanion.

FIG. 1(C) demonstrates the change of particle surface charge (zetapotential) during titration of DNA/PLL (1:3) particles with SPLL. Theparticle becomes negatively charged and accordingly recharged atapproximately the equivalence point (FIG. 1 (C)).

Thus, upon addition of large excess of non-decondensing polyanion smallnon-aggregated particles still exist, DNA is still condensed but thecharge of the particles becomes negative. We used atomic forcemicroscopy to visualize these negatively charged particles. FIG. 2 showssmall and non-aggregated 50 nm DNA/PLL/SPLL spheroids adsorbed on micain the presence of 1 mM NiCl₂.

Any water-soluble polyanion can be used for recharging purposesincluding succinylated PLL, succinylated PEI, polyglutamic acid,polyaspartic acid, polyacrylic acid, polymethacrylic acid, dextransulfate, heparin, hyaluronic acid, DNA, RNA, negatively chargedproteins, polyanions graft-copolymerized with hydrophilic polymer, andthe same carrying specific ligands.

Example 3

Stoichiometry of Purified Particles: To study the stoichiometry of therecharged complexes, DNA, PLL and SPLL polymers were labeled withrhodamine and fluorescein moieties to yield Rh-DNA, F1-PLL and F1-SPLLwith known degree of modification and adsorption coefficientsrespectively. Rh-DNA/F1-PLL/SPLL and Rh-DNA/PLL/F1-SPLL complexes wereformed in low salt buffer and then separated from non-boundpolyelectrolyte using density gradient ultracentrifugation.Corresponding amounts of each constituent can be determined by measuringoptical density at 495 nm and 595 mn respectively. DNA complexessediment through 10% sucrose solution and are retained in the separatinglayer between 10% sucrose and 40% metrizamide (metrizamide cushion). AllRh-DNA was found to be located on the sucrose/metrizamide border.Non-bound PLL and SPLL were found not to enter the 10% sucrose layer.DNA/PLL/SPLL complexes were found non-soluble and form precipitate onthe density layer. The recovered complexes were solubilized in 2.5 MNaCl and their visible spectra were analyzed. FIG. 3 representsRh-DNA/F1-PLL/SPLL (FIG. 3 a) and Rh-DNA/PLL/F1-SPLL (FIG. 3 b) complexspectra respectively together with standard Rh-DNA/F1-PLL andRh-DNA/F1-SPLL (1:1) charge ratio mixtures. The data clearly indicatesthat precipitated complex contains all three polyelectrolytes with astoichiometry of a 1:1:1 charge ratio.

Example 4

Zeta Potential of Purified Particles: As one may conclude fromstoichiometry studies, the DNA/PLL/SPLL (1:3:10) initial mixture alongwith 7× excess of free SPLL also contains 2× excess of PLL/SPLLparticles (“blank particles”) not complexing DNA. These particles werefound not to enter the 10% sucrose layer ensuring complete separation ofDNA containing particles from PLL and SPLL excess. Zeta potential wasmeasured using Brookhaven Instruments Corp. Zeta Plus Zeta PotentialAnalyzer. DNA concentration was 20 mg/ml in 1.5 ml of 25 mM HEPES, pH7.5.

Example 5

In vitro transfection enhancement upon recharging of DNA/polycationcomplexes. Recharging can increase the transfection activity ofDNA/polycation complexes. FIG. 4 shows the results of transfection ofHUH7 liver cells in 100% bovine serum with DNA/PEI (1:2 w/w) complexesrecharged with increasing amounts of SPLL (Mw=460 kDa). At optimal SPLLconcentration activity of recharged complex exceeds the activity of thenon-recharged one approximately 40 times. For transfection of rechargedcomplexes, 2 μg of the reporter plasmid pCILuc (expressing the fireflyluciferase cDNA from the human immediate early CMV promoter) (Zhang, G.,Vargo, D., Budker, V., Armstrong, N., Knechtle, S. & Wolff, J. HumanGene Therapy 8, 1763-1772 (1997)) was complexed with the polycation andpolyanion in low salt buffer. Resulting complexes were added to 35 mmwells containing cells at about 60% confluence. Transfected cells wereharvested 48 hours after transfection and cells were lysed and analyzedfor luciferase activity using a Lumat LB 9507 luminometer (EG&GBerthold).

Example 6

Recharged DNA/PEI complexes have reduced toxicity and exhibit genetransfer activity in vivo in an organism. Recharging of DNA/polycationcomplexes with strong polyanions which help to release DNA can also makecomplexes less toxic in vivo. Resulting complexes also are active ingene transfer in lungs upon i/v administration in mice. Table 1 showsthe toxicity of DNA/PEI/polyacrylic acid (PAA) complex is decreasingwith the increase of PAA content. Tertiary DNA/PEI/polyacrylic acidcomplexes were formed in 290 mM glucose, 5 mM HEPES, pH 7.4 at DNAconcentration of 0.2 mg/ml and PEI concentration of 0.4 mg/ml. Eachanimal was injected 0.25 ml of DNA complex solution. After 24 hours, theanimals were sacrificed, lungs, livers, hearts, kidneys were removed andhomogenized at 4° C. Luciferase activity of extracts (10 μl) wasmeasured using a Lumat LB 9507 luminometer (EG&G Berthold).

TABLE 1 In vivo gene transfer activity in mouse organs upon i/vadministration of DNA/PEI/PAA complexes (50 μg/100 μg). amount of PAAadded (μg) 40 50 60 70 Luciferase Activity, LU Liver 1465 3266 14537 387Lung 182187 9392 325 162335 Spleen 3752 1925 1647 1307 Heart 2186 158 761262 Animal 1/3 1/4 0/3 0/3 survival (dead/total)

Example 7

Crosslinking of polycation and polyanion layers on the DNA-containingparticles increases their stability in serum and on the cell surface.

Negatively charged (recharged) particles of condensed DNA can possessthe same physico-chemical properties as positively charged(non-recharged) ones. This includes flocculation in high salt solutions(including physiologic concentration). We found that chemicalcross-linking of cationic and anionic layers of the DNA particles cansubstantially improve stability of the particles in serum as well as onthe cell surface. Table 2 shows the time course of unimodal particlesize of DNA/PLL/SPLL crosslinked and non-crosslinked particles in 80%bovine serum as determined by dynamic light scattering.

TABLE 2 Particle sizing of DNA/PLL/SPLL crosslinked and non-crosslinkedcomplexes in 80% serum. Time, min crosslinking size (nm) no crosslinkingsize (nm) 0 153 104 15 154 105 60 171 108 200 246 115

Crosslinked particles essentially do not change their size in 200 min atroom temperature while non-crosslinked control flocculates rapidly.Crosslinking with cleavable reagents might help to overcome aninactivity problem. The polymers can also contain cleavable groupswithin themselves. When attached to the targeting group, cleavage leadsto reduce interaction between the complex and the receptor for thetargeting group. Cleavable groups include but are not restricted todisulfide bonds, diols, diazo bonds, ester bonds, sulfone bonds,acetals, ketals, enol ethers, enol esters, enamines and imines, acylhydrazones, and Schiff bases.

Example 8

Pegylation of polyanions for recharging. Recharging of DNA/polycationparticles with PEG-polyanion conjugates can substantially stabilizerecharged particles against salt-induced flocculation. Preparation ofPEG-SPLL conjugate. Water-soluble carbodiimide (EDC, 5 mg,) andN-hydroxysulfosuccinimide (S-NHS, 10 mg) were added to the 0.25 mlsolution of SPLL (20 mg/ml, Mw=210 kDa) at pH 5.0 and incubated for 5min at room temperature. Monoamino-polyethyleneglycol (4 mg, 0.4 ml in0.1 M HEPES, pH 8.0) was added to the SPLL and the mixture was continuedto incubate for 1 more hour. PEG-SPLL conjugate was dialyzed againstdeionized water overnight at 4° C. and freeze-dried. This preparationresulted in 5% (mol) substitution of COOH groups with PEG chains.

DNA-containing particles were prepared using the procedure in Example 1with the exception that SPLL-PEG conjugate was doubled compared to SPLL.Table 3 shows the time course of unimodal particle size of DNA/PLL/SPLLand DNA/PLL/PEG-SPLL particles in 80% bovine serum as determined bydynamic light scattering. Pegylated particles exhibit higher stabilitytowards flocculation as opposed to non-pegylated ones.

TABLE 3 Particle sizing of DNA/PLL/polyanion complexes recharged withSPLL and PEG-SPLL in 80% serum. Time, min Size (nm) SPLL Size (nm)PEG-SPLL 0 441 118 15 750 118 60 2466 139 120 5494 116

The foregoing is considered as illustrative only of the principles ofthe invention. Further, since numerous modifications and changes willreadily occur to those skilled in the art, it is not desired to limitthe invention to the exact construction and operation shown anddescribed. Therefore, all suitable modifications and equivalents fallwithin the scope of the invention.

1 A process for delivering a nucleic acid to a cell in vivo, comprising:a) forming a composition consisting of a nucleic acid associated via anon-covalent ionic interaction with a polycation in a solution whereinthe composition has a net charge less negative than the nucleic acid; b)ionically associating a polyanion with the composition of step a) insufficient amount to form a complex having a net negative charge; c)inserting the complex into a mammal; d) delivering the complex to thecell.
 2. The process of claim 1 wherein the polycation is selected fromthe group consisting of polylysine and polyethylenimine.
 3. The processof claim 1 wherein the polyanion comprises a molecule selected from thegroup consisting of succinylated PLL, succinylated PEI, polyglutamicacid, polyaspartic acid, polyacrylic acid, polymethacrylic acid, dextransulfate, heparin, hyaluronic acid, DNA, RNA, and negatively chargedproteins.
 4. The process of claim 1 wherein the polyanion comprises ablock co-polymer.
 5. The process of claim 1 wherein the polyanioncomprises a molecule selected from the group consisting of pegylatedderivatives, pegylated derivatives carrying specific ligands, blockcopolymers, graft copolymers and hydrophilic polymers.